6 methyl 16 oxy delta4 pregnenes and delta1, 4 pregnadienes



6 METHYL 16 OXY A PREGNENES AND A PREGNADIENES Frank H. Lincoln, Kalamazoo, William P. Schneider, Kalamazoo Township, Kalamazoo County, and Oldrich K. Sebek and George B. Spero, Kalamazoo, Mich, assignors to The Upjohn Company, Kalamazoo, Mich, a corporation of Michigan tes No Drawing. Application February 19, 1958 seri l No. 716,037

19 Claims. :(Cl. 260-69145) The present invention relates to steroid compounds and is more particularly concerned with 6a-methyl-11fi,16a, 17a-trihydroXy-4-pregnene-3,20-dione, 6a-rnethyl-9u-fiuoro-l1fl,16o,17a-trihydroxy 4 pregnene-3,20-dione, 618-, methyl 1l,B,l6oi,l7a trihydroxy-1,4-pregnadiene-3,20- dione, 6a-methyl-9a-fluoro-115,16a,'l7u-trihydroxy-1,4- pregnadiene-3,20-dione, the 16-esters thereof, the ll-keto analogues and the 16-esters thereof, and a method for the production thereof.

The novel compounds of this invention are illustratively represented by the following formula:

ene radical and R is selected from the group consisting of hydrogen and the acyl radical of an organic carboxylic acid, preferably a hydrocarbon carboxylic acid containing from one to twelve carbon atoms, inclusive.

The new compounds 6a-methyl-11fl,16u,17u-trihydroxy-4-pregnene-3,ZO-dione, 6a-methyl 9a fiuoro-llfi, l6a,l7u-trihydroxy 4 pregnene-3,20-dione, 6a-methy1- 11/8,160t,170t trihydroxy-l,4-pregnadiene-3,ZO-dione, 6amethyl 9a fluoro 11f3,16a,170t trihydroxy-lA-pregnadiene-3,20-dione, their 16-esters and the ll-keto analogues and the esters thereof, are highly active adrenocortical hormones having greater glucocorticoid and antiinflammatory activity than hydrocortisone or cortisone. In addition these compounds have diuretic activity and have salt-losing properties which make them especially well suited in the management of chronic congestiveheart failure and in the treatment of cirrhosis of the liver, the nephrotic and adrenogenital syndromes and the treatment of eclampsia and preclampsia. J i

The novel 6a-methyl-115,16a,17a-trihydroXyF4-pregatent ice nene-3,20-dione, 6a-methyl-9a-fiuoro11} ,16o ,'17a trihydroXy-4-pregnene-3 ,20-dione, 6a-methyl-l13, 16oz, 17a-t1ihydroxy-1,4-pregnadiene-3,20-dione, 6a-methyl-9 t-fiuoro 1 15,1618,17a-trihydroxy-1,4-pregnadiene-3,20-dione, the 16-esters thereof, the ll-keto analogues and '16-esters thereof can be given in oral, parenteral or topical compositions. The compounds can be administeredto the animal organism in conventional dosage forms suchas pills, tablets and capsules for oral use .or in conventional liquid forms as are used with natural and synthetic cortical steroid hormones for injection use. For topical use they can be administered in the form of ointments", creams, lotions and the like with or without CO-aicting' antibiotics, germicides and the like.

The process of the present invention comprises microbiological hydroxylation of 6u-methyl-l 15,17a-dihYdIQ2tY 4-pregnene-3,20-dione, 6a-methyl-9a-flu0ro 1l,B,l7a-d ihydroxy-4-pregnene-3,20-dione, and the A -analogues thereof to produce the corresponding 16-hydroxy compounds. Esterification of the thus produced 16-hydroxylated compounds is productive of the .16-esters. The corresponding 3 ll-keto analogues of these 1 6-hydroxy1ated compounds are obtained by oxidation of the ll-hydroxyl group of the above l6-esterified compounds with an oxidation agent such as chromic acid. If the free alcohols ofjth l'l-keto compounds are desired, the additional step of hydrolyzing the 16-esters such as with an alkali metal base is necessary. f

Starting materials for the present invention are 6amethyl-1l/3,17a-dihydroxy 4 pregnene-3,20,-ifdione 6pcmethyl-9oc-fiuoro 115,17 dihydroxy-4 pregnene-3,20- dione, 6a-methyl-l l,8,l7a-dihydroxy 1,,4Rjpregnadiene- 3 ,20-dione, and 6a-methyl-9 -fiuoro-1 lB,'17a-dihydroxye 1,4-pregnadiene-3,ZO-dione, prepared, as shown in the preparations.

In the bioconversion step of the present invention, the operational conditions and reaction procedure and details may be those already known in the art of steroid bioconversion as illustrated by the Murray et al. U. S. Patent 2,602,769, issued July 8, 1952, utilizing however the action of an organism of the'genus Streptomyc'es. Among the species which are useful in the fermentation of steroids are Streptomyce's roseochromogenus (Waksman collection 3689), Streptomyces sp. (A. T. C. C. 11009), and Streptomyces rose'ochromogenus (A. T. C. C. 3347).

The selected species of actinomycete is grown on a medium suitably containing. assimilable non-steroidal carbon, 'illustratively carbohydrates, such as dextrose; assimilable nitrogen, illustrativ'ely solubleor insoluble proteins, peptones or amino acids; and mineral constituents, illustratively sodium or ammonium phosphate and magnesium sulfate. The medium may desirably have .a pH before inoculation of between about 6.5 to about 7.8 .thougha higher or, lower pH may be used. A pH of between about 6.8 and about 7.4 is preferred for the growth of actinomycetes and a temperature range from about 20 to about 35, degrees centigrade with about 20 to 32 degrees centigrade preferred. I

The growth period required before the steroid to be fermented is exposed to the actinomycetedoes notappear to be critical, for example, the steroid may be added either before sterilization of the medium, at the time of inoculating the medium or at sometime later, for e iample, 24 to 48 hours later. The addition of steroidsl bstrate to be fermented may be accomplished in any suitable manner, such as by dispersing the steroid substrate, either alone with a dispersing agent, or in solution in an organic solvent. Either submerged or surface culture procedures may be used with facility, although submerged culture is preferred.

The temperature during the period .of fermentation of the steroid may be the same as that found suitable for the growth of the organism. It need be maintained only within such range as supports life, active growth, or the enzyme activity of the streptomycete. The time required for the fermentation of steroid varies somewhat with the procedure. When the steroid is added to the actinornycete after substantial growth of the organism, for example, after sixteen to 24 hours at optimum temperature, the conversion of steroid substrate begins immediately and is substantially complete in from two to ten days, five days being generally satisfactory.

After completion of the steroid fermentation, the resulting transformed steroid is recovered from the fermentation reaction mixture by extracting the fermentation reaction mixture, including the fermentation liquor and mycelium with an organic solvent for steroids, for ex ample, methyl isopropyl ketone, methylene chloride, chloroform, carbon tetrachloride, ethylene chloride, trichloroethylene, ether, amyl acetate, benzene, and the like. The fermentation liquor and mycelium can be separated and then separately extracted with suitable solvents. The extracts can be combined, either before or after washing with an alkaline solution, illustratively sodium bicarbonateQsuitably dried, as for example, over anhydrous sodium sulfate, and the resulting purified transformed steroid obtained by recrystallization from organic solvents, by trituration or by chromatography in order to isolate the thus obtained steroids from the other fermentation products.

Bioconversion of 6a-methyl-l1B,17a-dihydroxy-4-pregnene-3,20-dione, 6m-methyl-9a-fluoro-11fl,l7u-dihydroxy- 4-pregnene-3,20-dione, 6a-methyl-1 1fi,l7a-dihydroxy-1,4- pregnadiene-3,20-dione, and 6a-methyl-9 x-fluoro-l113,170:- dihydroxy-l,4-pregnadiene-3,20-dione according to the fermentation procedure above described, is productive of 6a-methyl-1l,B,16a,17utrihydroxy 4 pregnene-3,20- dione, 6a methyl 9a fiuoro-l1B,16a,17a-trihydroxy-4- pregnene-3,20-dione, 6a-methyl-1 1,8,1 6a,l7a-trihydroxy- 1,4 pregnadiene-3,20-dione, and 6u-methyl-9a-fiuoro-llfl, 16a,17a-trihydroxy-1,4-pregnadiene 3,20 dione, respectively.

The l6-hydroxylated compounds thus produced can be esterified to produce the corresponding 16-esters. This reaction can be performed under esterification conditions known in the art, e. g., by the reaction of the hydroxy compound with the selected acid halide, e. g., acid chloride or acid bromide, the anhydride of a hydrocarbon carboxylic acid, or by reaction with the selected acid, in the presence of an esterification catalyst or with an ester under ester exchange reaction conditions. Reaction conditions which are apt to affect the labile 11 8-hydroxy group should be avoided. Compounds thus produced include the l6-acyloxy compounds represented by Formula I wherein X is p-hydroxymethylene radical and wherein R is the acyl radical of an organic carboxylic acid, preferably a hydrocarbon carboxylic acid containing from one to twelve carbon atoms, inclusive, e. g., formic, propionic, butyric, isobutyric, valeric, isovaleric, trimethylacetic, Z-methylbutyric, B-ethylbutyric, hcxanoic, diethylacetic, triethylacetic, heptanoic, octanoic, a-ethylisovaleric, succinic, a cyclic acid, e. g., cyclopropylideneacetic, cyclopentylformic, cyclopentylacetic, B-cyclohexylpropionic, cyclohexylformic, cyclohexylacetic, an aryl or alkaryl acid, e. g., benzoic, 2-, 3- or 4-methylbenzoic, 2,3-, 2,4-, 2,5-, 2,6-, 3,4- and 3,5-dimethylbenzoic, ethylbenzoic, 2,4,6-trimethylbenzoic, 2,4,6-triethylbenzoic, a-naphthoic, 3 methyl a naphthoic, an aralkyl acid, e. g., phenylacetic, phenylpropionic, diphenylacetic, triphenylacetic, an unsaturated acid, e. g., acrylic, maleic, vinyl acetic, propiolic, undecolic, etc. Illustrative of the esters thus produced are the 16-acylates such as 6a-methyl-l118,1601,17a-trihydroxy-4-pregnene-3,20-dione 16-acetate, a-methyl-l1;8,16a,17x-trihydroxy, 1,4 pregnadiene-3,20-dionc 16 acetate, 60:-

methyl-9a-fiuoro-11;3,16a,l7a trihydroxy 4 pregnene- 3,20-dione 16-acetate, 60C-[I1Cthyi-9a-flUOIO'11fl,160t,170ttrihydroxy-l,4-pregnadiene-3,ZO-dione l6-acetate, and the like.

The llfi-hydroxyl of the thus produced 16-acylates can be oxidized to the corresponding ll-ketone with an oxidizing agent. Oxidizing agents such as chromic acid, potassium dichromate, a halo-amide, and the like are operative. The oxidation can be carried out by a variety of methods, such as, for example, by oxidizing the said 11fi-hydroxy steroid in acetic acid-water solution with chromium trioxide, using molar quantities or a slight excess, such as from ten to thirty percent excess, or by oxidizing with a haloamide or imide of an acid, such as N-bromoacetamide, N-chlorosuccinimide, or N-brornosuccinimide dissolved in pyridine, dioxane, or other suitable solvents. At the conclusion of the desired oxidation reaction, the excess oxidant is generally destroyed by addition of methyl alcohol, ethyl alcohol, and the like for the chromic acid oxidant or a bisulfite for N-bromoacetamide, N-bromosuccinimide and other N-haloacylamides and imides. Thereafter, the resulting ll-keto product is recovered by conventional means, such as by dilution with water and extraction with a water-immiscible solvent, e. g., methylene chloride, ether, benzene toluene, ethyl acetate, or the like. Illustrative of the 11- keto l6-acylates thus produced are for example, 601.- methyl-l6a,l7a-dihydroxy-4-pregnene 3,11,20 trione l6 acetate, 60c methyl-9a-fiuoro-16a ,17a-dihydroxy-4- pregnene-3,ll,20-trione lG-acetate, 6u-methyl-l6a,l7adihydroxy-1,4-pregnadiene-3,l1,20-trione l6-acetate, 60cmethyl-9a-fluoro-16a,17a.-dihydroxy 1,4 pregnadiene- 3,11,20-trione 16-acetate, and the like.

The ll-keto 16-acylates thus produced may, if desired, be converted to the free alcohols, i. e., 6a-mthyl-16oc,17adihydroxy-4-pregnene-3,1 1,20-trione, 6a-1I16thyl-9a-flllO- ro-16a,17a-dihydroxy-4-pregnene-3,l1,20-trione, 6a-methyl-16a,l7a-dihydroxy-1,4-pregnadiene-3,1 1,20-trione and 60a methyl 9a fluoro 1611,1711 dihydroxy 1,4 pregnadiene-3,11,20-trione. The alcohols are obtained from the acylates by hydrolysis in accordance with general hydrolysis procedures known in the art. A preferred procedure is to employ at least a molar equivalent of an alkali-metal bicarbonate in a substantially oxygen-free solution of a mixture of a lower alkanol and water. The hydrolysis reaction is carried out at a temperature between ten and thirty degrees centigrade While protecting the mixture from atmospheric oxygen. After the hydrolysis is complete, the reaction mixture is neutralized with an acid, e. g., acetic acid, and the hydrolyzed product recovered from the reaction mixture by evaporation and crystallization, extraction with methylene chloride, or the like. The l6-esters when desired, can again be prepared by esterification of the hydroxyls by esterification procedures hereinbefore described. The preferred esters are those derived from an organic carboxylic acid, preferably a hydrocarbon carboxylic acid containing from one to twelve carbon atoms, inclusive.

The following preparations and examples are illustrative of the process and products of the present invention, but are not to be construed as limiting.

PREPARATION 1 6a-methyl-11;3,I 7a-dihydroxy-4-pregnene-3,20-dione A. 6:: M'ETHYL 11B,17a.21 TRIHYDROXY 4 PREG- NENE 3,20 DIONE 21 METHANESULFONATE (6a METHYLHYDROCORTISONE 21 "\IETHANESUL- FONATE) A solution was prepared containing one gram (2.65 millimoles) of 6a-methylhydrocortisone [Spero et. al., I. Am. Chem. Soc. 78, 6213 (1956)], in seven milliliters of pyridine. This solution was cooled to zero degrees centigrade and treated with 0.3 milliliter of methanesulfonyl chloride. Thereafter the solution was allowed to stand at zero to five degrees centigrade for a period of 5 two hours. The reaction mixture was then diluted with water and extracted wlth three 25-milliliter portions of methylene chlpride. The extracts were combined, washed with egld dilute hydrochlorlc acid (until a pH of two to three was maintained in the aqueous layer), then washed again with cold sodium bicarbonate solution, water, and finally dried over anhydrous sgdium sulfate. Evaporation of the methylene chloride extract at reduced pressure gave a white glassy product of 6a-methyl-l 1,8,17u,21- trihydroxy-4-pregnene 3,20-dione ZI-methanesulfonate.

B. (in METHYL 1113,17 1 DIHYVDROXY 21 IODO 4- PREGNENE3,20-DIONE The crude methanesulfonate was dissolved in fifteen milliliters of acetone and treated with a solution of one gram of sodium iodide in ten milliliters of acetone. The mixture was heated under reflux with stirring for a period of fifteen minutes, the heat was then reduced and the mixture concentrated to dryness at reduced pressure to give a-mcthyl-l1B,l7a-dihydroxy+21riodor4-pregnene-3, 20-dione.

9 34 3.1 5. 1 115 .17.9 H PBQKF? 4 BEGNEN 3,20-DIONE The ude fist-ms ll -l 5 1 ah droxy-2 dm p esns -fifiQ-d oue a o ain d et ewes s u i d w fifteen milliliters of acetic acid and stirred for a period of 45 minutes. Thereafter one gram of zinc dust was added and stirring continued for fifteen minutes. The excess zinc dust was removed by filtration, the filtrate a di ute w meth len chlo ide and ashed ith water and cold sodium hiearbonate solution until all acetic acid was neutralized. After drying over anhydrous sodium sulfate, the solution was ehromatographed over 75 grams of Florisil synthetic magnesium silicate taking fractions of 200 milliliters each as follows: fourteen fractions of Skellysolve B hexanes containing ten percent acetone and one fraction of 10.0 percent acetone.

Fractions eight through thirteen, inclusive, were combined and evaporated to give 760 milligrams of crystals which after recrystallization from acetone-Skellysolve B hexanes yielded in two crops 650 milligrams of 6u-methyl- 11,6,17a-dihydroxy-4-pregnene-3,ZO-dione of melting point 203-210 degrees centigrade.

AnalysisTCalcd. for C H O C, 73.30; H, 8.95. Found: C, 73.19; H, 8.59.

PREPARATlON 2 6ametl 1 yl-1 113,1 7ix-dihydr0xy-1 ,4-pregna di e ne- 3,20Egiione A- 55 I E JP 141 17 1?! @BIHYDBQ 1 P EG ADI E 3IZQ D Zl-METHANE QFQNATE A solution was prepared containing 16.4 milligrams (0.437 millirnole) .Of '1-dehydro-6:methylhydrocortisone [Spero et 3. J. Am. Chem. Soc. 78, 6213 (1956)], in one milliliter of pyridine. This solution was cooled to zero degrees eentigrade and treated with a cooled solution of 75 milligrams of methanesulfonyl chloride in 0.5 milliliter of pyridine. Thereafter the solution was al+ lowed to stand at a temperature between zero and five ee Centi ade for a Pe i d f tw hours. Thereafter ice and sutlicient dilute hydrochloric acid to neutralize P id ne was added and the mixture extracted with three 25-milliliter portions of methylene chloride. The extracts were combined, washed with cold sodium bicarbonate solution, then water and finally dried over anhydrous sodium sulfate and evaporated at reduced pressure to give crystalline 6u-methyl11,8,17o ,21-trihydroxy-1,4- preg adiene-3,20sd ione ZI-methanesulfonate.

B. 601 METHYL 115,171 DIHYDROXY 21 10130 1,4-

' PREGNADIENE-3,20-DIONE The crystalline methanesulfonate of 6a-methyl-11,17a, 21-trihydroxy-1,4-pregnadiene-3,ZO-dione from above was dissolved in fifteen milliliters of acetone and treated witha solution of 200 milligrams of sodium iodide in five 6 milliliters of acetone. The mixture was heated under reflux with stirring fora period of fifteen minutes. The heating was then discontinued and the mixture concen tratedto dryness at reduced pressure to give G -methyl- 1118,17a-dihydroxy-2l-iodo-1,4-pregnadiene-3,20-diene.

C. 611: METHYL 11,13,170. DIHYpRoXY -14 PBEQNA:

DIENE-3,20-DIONE The crude 6u-methyl-l1g,17a-dihydroxy-21-iodo-1,4- pregnadiene-3,20-dione was slurried with five milliliters of acetic acid and stirred for a period of 45 minutes. Thereafter was added an aqueous solution of 250 milligrams of sodium thiosulfate pentahydrate causing the iodine color to disappear. Additional water was added (fi mi il ters) and he mixture extracted ith thre 25-milliliter portions of methylene chloride. The methylene chloride extracts were combined, washed with water and cold sodium bicarbonate solution until all acetic acid was neutralized. After drying over anhydrous sodium sulfate, the solution was concentrated to approximately fifteen milliliters and chromatographed over ten grams of Florisil (synthetic magnesium silicate) taking fractions of fifty milliliters each as follows: nine fractions of Skellysolve B hexanes containing ten percent acetone, four fractions of Skellysolve B hexanes containing'fifteen percent acetone and one fraction of 100 percent acetone.

Fractions six through eleven, inclusive, were combined 1 and evaporated to give 136 milligrams of crystals which after recrystallization from acetone-Skellysolve B hexanes yielded milligrams (72 percent overall yield) of 6amethyL-l 1B, l7a-dihy,droxy=1,4=pregnadienew3,20-dione of me in 222 t9 2 de e c n Analysis.--Calcd. for C H Q C, 73.71; H, 8.44. Found: C, 73.30; H, 8.44.

PREPARATION 6oz methyl 9a fluoro 1118,1711 dihydroxy 4 pregnene 3,20 dione A. Bar METHYL 9a FLUORO ill/ 9,170.31 TRIHY;

DROXY 4 PREGNENE 3,20 DIONE 21 METHANE- SULFONATE A solution was prepared containing one gram of 6amethyl-9a-fluorohydrocortisone [Spero et al., J. Chem. Soc. 79, 1515 (1957)], in ten milliliters of pyridine. This solution was cooled to zero degrees centigrade and treated with 0.5 milliliter of methanesnlfonylchloride. Thereafter the solution was allowed to stand at a temperature between zero and live degrees centigrade for a period of eighteen hours. Thereafter ice and ten milliliters of water was added followed by milliliters of suflicient dilute (five percent) hydrochloric acid to neutralize the pyridine. The mixture was then filtered, the precipitate thus collected, washed with water, and dried to give 1.2 grams of crude 6oa-methyl-9a-fluoro-11 3,17a,21- trihydroxy-4-pregnene-3,20-dione ZI-methanesulfonate of melting point 166 to 178 degrees centigrade (dec.).

B. 60: METHYL 9a. FTJUQRO 115,170! 1 )IHYDRox- 21-IODO-4-PREGNENE-3,20-DIONE 750 milligrams of crude methanesulfonate of 61x methyl 9a fluoro 11,8,17oz,2l trihydroxy 4 pregnene 3,20 dione was dissolved in fifteen milliliters of acetone and treated with a solution of 750 milligrams of sodium iodide in fifteen milliliters of acetone. The mixture was heated under reflux with stirring for a period of fifteen minutes. The heating was then discontinued and the mixture concentrated to dryness at reduced pressure to give 60: methyl 90c fluoro T 1116,1711; dihydroxy 21 iodo 4 pregnene 3,20 dione. l

c. 612 METHYL 9a FLUORO l1.B,17a-DIHYDROXY 4- PREGNENE3,20-DIONE The crude 60c methyl 9p; fluoro 11,8,17u dihydroxy 21 iodo 4 pregnene 3,20 dione was slurried with fifteen milliliters of acetic acid and stirred for a a. riod of 45 minutes. Thereafter 750 milligrams of zine dust was added and stirring continued for twenty minutes longer.. .Thereaction mixture was then filtered, the filtrate diluted with methylene chloride and washed with aqueous potassiuma-carbonate until neutral. The organic layer was separated; dried over sodium sulfate, concentrated to 25 milliliters and chromatogramed over forty grams of Florisil anhydrous magnesium silicate. The column was developed taking fractions of 100 milliliters each as follows:

TABLE 1 Fraction Solvent 1-15 Skellysolve B hexane-10% acetone. 16 acetone.

PREPARATION 4 6o: methyl 9a fluoro 115,170: dihydroxy 1,4 pregnadiene 3,20 dione A. 60: METHYL 9a FLUORO 11B,17a,21 TRIHY- DROXY 1,4 PREGNADIENE 3,20 DIONE 21- LHETHANESULFONATE A solution was prepared containing'250 milligrams of 1-dehydro-6a-methyl-9a-fluorohydrocortisone [Spcro et al., J. Am. Chem. Soc. 79, 1515 (1957)], in six milliliters of pyridine. This solution was cooled to zero degrees centigrade and treated with 0.25 milliliter of methanesulfonyl chloride. Thereafter the solution was allowed to stir at a temperature between zero and five degrees centigrade for a period of eighteen hours. Thereaftcr'ice and two milliliters of water were added, followed by thirty milliliters of sufficient dilute (five percent) hydrochloric acid to neutralize the pyridine. The mixture was then filtered, the precipitate washed with water, and dried to give 197 milligrams of crude 6a methyl 9a fluoro 1lfi,17a,21 trihydroxy 1,4 pregnadiene 3,20 dione ZI-methanesulfonate of melting point 165 to 185 degrees centigrade.

B. 60: METHYL 9a. FLUORO 118,171: DIHYDROXY- 21-IODO-1,4-PREGNADIENE-3,20-DIONE The crude 197 milligrams of methanesulfonate of 60a methyl 9oz fluoro 1l/3,17oz,2l trihydroxy 1,4 pregnadiene- 3,20 dione from above was dissolved in five milliliters of acetone and treated with a solution of 197 milligrams of sodium iodide in five milliliters of acetone. The mixture was heated under reflux with stirring for a period of fifteen minutes. The heating was then discontinued and the mixture concentrated to dryness at reduced pressure to give 60: methyl 90c fluoro 115,170; dihydroxy 21 iodo 1,4 pregnadiene 3,20 dione.

C. 8n METHYL 9a FLUORO 115,170. DIHYDROXY- 1,4-PREGNADIENE-3,20-DIONE milligrams of crude .6m-methyl-9a-fluoro-11,3,17 -dihydroxy-1,4-pregnadiene-3,20-dione.

, Thecrude material was then chromatographed by dissolving 120 milligrams of 6a-methyl-9a-fiuoro-l15,170:- dihydroxy-1,4-pregnadiene-3,ZO-dione in 300 milliliters of methylene chloride and allowing the thus obtained solution to be adsorbed on a chromatographic column containing ten grams-of Florisil synthetic magnesium silicate. The column was developed taking fractions of twenty milliliters each as follows:

TABLE II Fraction Solvent Skellysolve B hexane-% acetone. Skellysolve B hexane-% acetone.

. Skellysolve B hexane-% acetone. Skellysolve B hexane'20% acetone. Skellysolve B hexane-% acetone. Acetone.

Fractions 11 through 24 inclusive were combined, evaporated and twice recrystallized from acetone to give pure 6oz methyl 9a-fluoro-l 1fi,l7u-dihydroxy-1,4-pregnadiene-3,20-dione of melting point 292 to 303 degrees centigrade.

EXAMPLE 1 6a methyl 11B,16a,17a trihydroxy 4 pregnene 3,20-dione One hundred milliliters of two percent cornsteep liquor of sixty percent solids was adjusted to pH of 6.8 to 7.4 with sodium hydroxide and was sterilized at fifteen pounds pressure for thirty minutes. To this was added a similarly sterile solution of two grams of Cerelose (technical grade of dextrose) in four milliliters of water. This sterile medium was inoculated with a suspension of spores and mycelium of Streptomyces roseochramogenus (Waksman Collection No. 3689) and was agitated on a rotary shaker for a period of 24 hours by whichtime a good growth of the organism had taken place. To this 24 hour culture, twenty milligrams of 6a methyl 11,8,1711 dihydroxy 4 pregnene 3,20 dione dissolved in 0.2 milliliter of dimethylformamide was added. Incubation of the steroid with the microorganism was maintained (with agitation) for five days, at which time the pH was 8.6. The fermentation broth was then separated into the mycelium and the beer by centrifugation. The mycelium was extracted first with two 25 milliliter portions of acetone and then with four successive 25 milliliter portions of methyl isopropyl ketone. The beer was extracted with four successive 25 milliliter portions of methyl isopropyl ketone. All of the extracts were combined, washed with two percent aqueous sodium bicarbonate solution and with water, dried with anhydrous sodium sulfate, and evaporated to dryness. The residue, which on paper chromatogram analysis showed the presence of 6m-methyl-11/3,l6a,17atrihydroXy-4-pregnene-3,20-dione, was purified by chromatography over synthetic magnesium silicate (Florisil) and crystallization from acetone to give 6a-methyl-11 3,-

' 16a,17a-trihydroxy-4-pregnene-3,20-dione.

Following the procedure of Example 1 above, but substituting 6a methyl-9a-fluoro-11p,17a-dihydroxy-4-pregnene-3,20-dione, 6a-methyl-115,17u-dihydroxy-L4-pregnadiene-3,20-dione, or 6a-methy1-9a-fluoro-11B,l7u-dihydroxy-l,4-pregnadiene-3,ZO-dione as starting material therein, is productive of 6IZ-mEthYI-9CC-flUOI'O-I1B,16E(,17d' trihydroxy-4-pregnene-3,20-dione, 6a. methyl 11 3,1601,- 17a-trihydroxy-1,4-pregnadiene-3,20-dione, and 6a-methyl fluoro 1113,16ot,17a trihydroxy 4 pregnene-3,20-dione, respectively.

. ,9 EXAMPLE 2 6o; methyl 115160 170; trihydroqcy 4 pregnene 3,20-dione Iii-acetate A solution of 1.2 grams of 6a-methyl-1lfl,16oz,17mtrihydroxy-4-pregnene-3,ZO-dione (from Example 1) in twenty milliliters of pyridine and twenty milliliters of acetic anhydride was allowed to stand at room temperature (about 25 degrees centigrade) for eighteen hours and was then poured into 200 milliliters of ice-water. The resulting mixture was extracted with methylene chloride and the extract was washed with dilute hydrochloric acid, dilute sodium bicarbonate, and water. After drying the solution with anhydrous sodium sulfate, the solvent was removed by evaporation and the residue was purified by chromatography over synthetic magnesium silicate (Florisil) and crystallization from acetone to give 6a-methyL-11,3,16 ,17a-trihydroxy-4-pregnene-3,20-dione 16-acetate.

Following the procedure of Example 2 above but substituting 6a methyl-9a-fiuoro-11p,16a,17a-trihydroxy-4- pregnene-3,20-dione, 6a-methyl-11fl,16u,17a-trihydroxy- 1,4-pregnadiene-3,20-dione or 6a-methyl-9a-fluoro-1lfl,- 1. 1h t ih drqx -lAr sna n fi z k s as Starting material therein is productive of .6 a-methyl.- 9u.-fluoro.- 1 1 3,16 ,17 trihydroxy 4-pregnene-3 ,20-.dione 16-ace tate, 6 g-methyl-l1 8,16a,17atrihydroxyr1,4-pregnadiene- 29=i91. a p awta and a-methyl- -flu ro- 11 6 lh -t ib mxylArmen dienw Z di Q 16-3 m, e sst va y- Similarly, acylation of 6a methyl 1 1,1 6 ;,1 7 tr ihyd aty esume .20; di ne th the ppr pri acid arihydride or acid chloride is produptive of still other 16 acylates such as, for example, 611 methyla- 1lfi,16oc, 17a trihydroxy 4 pregnene 3,20 dione 16 propimrate, 6. meth l 5 1 r hydr x 4 pr nene 3,20 dione 16 butyrate, 6 methyl 115,16q, 1705 trihydroxy .4 preg-nene 3,20 dione 16 valerate, 6a methyl 115,16a,17a trihydroxy 4 pregnene- 3,20 dione 16 7 hexanoate, 6a methyl 11j3,16o &,170ztrihydroxy 4 pregnene 3,2 0 1 dione 16 7 laurate, 6a,- methyl 11346 1470; trihydroxy 4 pregnene 3,20- dione 16 (0-, m-, p-oluate), 6a methyl 1 113,16 ,170- lrih sl aay 4 u asnen ,2 dione 1 b yra 6a methyl 1153,16gg17a trihydroxy 4 pregnene- 3,20 dione 16 isovalerate, 6a methyl 11,8,16a,17atrihydroxy 4 pregnene 3,20 dione 16 cyclohexane carboxylate, 6a methyl ll/3,l6oc,17a trihydroxy 4- pregnene 3,20 dione 16 benzoate, 6a methyl- 11,8,16a,17a trihydroxy 4 pregnene 3,20 dione 16- phenylacetate, 60c methyl 1lfi,16a,17a trihydroxy 4- pregnene 3,20 dione 16(,8 phenyl) propionate, 6amethyl 11,8,16u,17a trihydroxy 4 pregnene 3,20- dione 16 0-, m-, p-tolu'ate), 6a methyl 11,8,16a,l7oz trihydroxy 4 pregnene 3,20 dione 16 hemisuccinate, 6a methyl 11,8,16a,l7o trihydroxy 4 pregnene- 3,20 dione 16 hemiadipate, 60c methyl 11,8,16a,17atrihydroxy 4 pregnene 3,20 dione 16 acrylate, 6v.- methyl l1B,16oc,l7oc trihydroxy 4 pregnene 3,20- dione 16 undecylenate, 6a methyl 11,8,16u,17a trihydroxy 4 pregnene 3,20 dione 16 propiolate, 6a methyl l1 3,16oc,l7a trihydroxy 4 -pregnene- 3,20 dione 16 cinnamate, 60c methyl 115,16a,17octrihydroxy 4 pregnene 3,20 dione 16 maleate, 6amethyl l1;8,16e,l7u trihydroxy 4 pregnene 3,20- dione 16 citraconate, and the like.

Similarly, acylation of 6a methyl 90c fluoro- 11fi,16oc,17ct trihydroxy 4 pregnene 3,20 dione, 60c methyl 11 B,16a,17oa trihydroxy 1,4 pregnadiene- 3,20 dione, or 60; methyl 90c flu0I0-11fi,16oc,17oc trihydroxy 1,4 pregnadiene 3,20 dione with the appropriate acylating agent is productive of the corresponding 16 acylates. The preferred acylates are those corresponding to the acylates described above for 60 methyl- 11/3,16a,17a trihydroxy 4 pregnene 3,20 dione.

' 10 EXAMPLE 3 6a-methyl-I 6 a,] 7a-dihydr0xy-4-pregnene-3,11,20-tri0ne 16-acetate To a solution of 0.5 gram of 6a methyl 1lfi,l6a,l7octrihydroxy 4 pregnene 3,20 dione 16 acetate in twenty milliliters ,of acetic acid was added a solution of 0.15 gram of chromium trioxide in one-half milliliter of water. The mixture was stirred and maintained at room temperature (about 25 degrees centigrade) for a period of four hours. Thereafter the excess oxidant was destroyed by the addition of 0,5 milliliter of methanol and the mixture was poured into milliliters of water and extracted with methylene chloride. The extract was a he h ut s dium b arbonate and w h wate and was dried and evaporated to dryness. The residue a s s l d f om t n o s 6a me h 16, 17 edih dmx p sa LZ -t Q a 1 -week.-

Follcf i a the P o ion of Examp 3 a o but uib= tun n 9 me y 9 u r leum? t hydroxy 4 pregnene 3,20 dione 16 acetate, 6pcrnethyl llfi,l6ca,l704 trihydroxy 1,4 pregnadiene? 3,20 dione l6 acetate, or 600 methyl 9a fluorol1,B,160c,17oc trihydroxy 1,4 pregnadiene 3,20 dione 16 acetate as starting material therein is productive of 6a methyl 9a fiuor'o T 1604,1704 dihydroxy 4 pregnene 3,11,20 trione 16 acetate, 6m methyl 1601,17otdihydroxy 1,4 pregnadiene 3,11,20 trione 16- acetate, and 6a methyl 9a fluoro 16a,17a dihydroxy 1,4 pregnadiene 3,11,20 trione 16 acetate, respectively.

Similarly oxidation of other 11,8 hydroxy 16 ester compounds of Example 2 is productive of the corresponding 11 keto 16 ester compounds. The preferred l1- keto 16 ester compounds are those wherein the acyl radical is that of an organic carboxylic acid, preferably a hydrocarbon carboxylic acid containing from one to twelve carbon atoms, inclusive.

EXAMPLE 4 6qc-methyl-16m1 7oc-d ihydroxy-4-pregnene-3 ,1 1,20-tri0ne gen and stirred at 25 degrees centrigrade for eight hours.

The solution was then neutralized by addition of acetic acid and the methanol was removed by distillation under reduced pressure. The residue was extracted with 100 milliliters of methylene dichloride and the extract, after drying over sodium sulfate, was chromatographed over a column of eighty grams of synthetic magnesium silicate using Skellysolve B hexanes with increasing amount of acetone for elution. The product fraction from the column was crystallized from acetone to give 60c methyl- 16a,17a dihydroxy 4 pregnene 3,11,20 trione.

Following the procedure of Example 4, saponification of Get methyl 90c fluoro :,17m dihydroxy 4- pregnene 3,11,20 trione 16 acetate, 6a methyl- 16a,l7a dihydroxy 1,4 pregnadiene 3,11,20 trione 16 acetate and 60a methyl 90c fluoro 16a,17a dihydroxy 1,4 pregnadiene 3,11,20 trione 16 acetate of Example 3 is productive of the corresponding hydroxy compounds, 60c methyl 9a fluoro 1604,170; dihydroxy 4 pregnene 3,11,20 trione, 6o: methyl- 16a,l7a dihydroxy 1,4 pregnadiene 3,11,20 trione and 604 methyl 9oz luoro 1600,1700 dihydroxy 1,4- pregnadiene 3,11,20 trione.

It is to be understood that the invention is not to be limited to the exact details of operation or exact compounds shown and described, as obvious modifications and equivalents will be apparent to one skilled in the art, and the invention is therefore to *be limited only by the scope of the appended claims.

v11 We claim: 1. A l6-oxygenated steroid of the formula:

C=O CHa IMOH -OR Y wherein the 1,2-carbon atom linkage is selected from the linkages consisting of single bond and double bond linkages, Y is selected from the group consisting of hydrogen and fluorine, X is selected from the group consisting of the carbonyl radical and the fi-hydroxymethylene radical, and R is selected from the group consisting of hydrogen and the acyl radical of a hydrocarbon carboxylic acid containing from one to twelve carbon atoms, inclusive.

2. A 16-oxygenated pregnene of the formula:

C=O OH: I... 011

wherein Y is selected from the group consisting of hydrogen and fluorine, X is selected from the group consisting of the carbonyl radical and the fl-hydroxymethylene radical, and R is selected from the group consisting of hydrogen and the acyl radical of a hydrocarbon carboxylic acid containing from one to twelve carbon atoms, inelusive.

3. 6a methyl l1}9,l6oc,17oc trihydroxy 4 pregnene-3,20-dione.

4. 6c: methyl 9a fluoro 11;3,l6u,17a trihydroxy- 4-pregnene-3,20-dione.

5. 6a methyl 16a,17a dihydroxy 4 pregnene- 3,11,20-trione.

pregnene-3,l 1,20-trione. r

6. 6a methyl 9a fluoro 160:,170: dihydroxy 4- 7. 6oz methyl- 11fl,l6a,17a trihydroxy l 4 preg nene-3 .20-dione 16-acetate.

8. 6a methyl 9a fluoro 11B,16u,17a trihydroxy- 4-pregnene-'3,20-dione 16-acetate.

9. 6a methyl 16ct,17a dihydroxy 4 pregnene- 3,11,20-trione l6-acetate.

. 10. 6oz methyl c fluoro :,1711 dihydroxy 4- pregnene 3,11,20-trione l6-acetate.

11. A l6-oxygenated pregnadiene of the formula:

'wherein Y is selected from the group consisting of hydrogen and fluorine, X is selected from the group consisting of the carbonyl radical and the fi-hydroxymethylene radical, and R is selected from the group consisting ofhydrodroxy-1,4-pregnadiene-3,ZO-dione l6-acetate.

18. 6oz methyl 16cz,17u dihydroxy 1,4-pregna- 'diene-3,l 1,20-trione 16-acetate.

19. 6oz methyl 9oz fluoro-16,17a dihydroxy- 1,4-pregnadiene-3,11,20-trione 16-acetate.

No references cited.

UNITED STATES PATENT OFFICE Certificate of Correction Patent No. 2,864,838 December 16, 1958 Frank H. Lincoln et a1.

It is hereby certified that error appears in the printed specification of the above numbered patent requiring correction and that the said Letters Patent should read as corrected below.

Column 1, lines 47 to 50, the illustration of the fl-hydroxymethylene radical should appear as shown below instead of as in the patentcolumn 9, line 43, for 16-(o, m-, p-o1uate), read 16-trimethylacetate,--.

Signed and sealed this 30th day of June 1959.

Attest:

KARL H. AXLINE, ROBERT C. WATSON,

Attesting Ofiaer. 1 oonwm'ssioner of Patents. 

1. A 16-OXYGENATED STEROID OF THE FORMULA: 